BTEC Level 3 Applied/Forensic Science
Unit 2: Practical Scientific Procedures and Techniques
Learning Aim C: Undertake chromatographic techniques to identify components in mixtures
Matthew Howard (9360)
May 2022
Introduction: What is chromatography?
Chromatography can be described as the process of separating components into a mixture. To get
this process started, the mixture is dissolved into a substance called the mobile phase, this then
carries it towards the second substance which is called the stationary phase. The mobile phase may
be either a liquid or a gas, while the stationary phase is either a solid or a liquid. Different substances
will travel through the stationary phase at different speeds, this will then cause it to separate from
one another, this is how the results of chromatography are shown and collected. The travel times of
the substances is called the retention time which is used to find out the results of the experiment. By
altering the mobile phase and the stationary phase a wide variety of chromatographic methods have
been created, each serving a different purpose and ideal for different mixtures. Some of the most
common forms of chromatography are paper chromatography, thin layer chromatography, liquid
chromatography and gas chromatography.
What is paper chromatography?
Paper chromatography is an analytical method used to separate coloured substances by taking
advantage of their different rates of migration across sheets of paper. This chromatography
technique is most commonly used in a school setting now but it has been replaced in the laboratory
setting by thin layer chromatography, this is because it shows a greater separation of the
components in the mixture which makes it much easier to analyse. The method consists of applying
the test solution as a spot near one corner of a sheet of filter paper, the paper is initially applied with
a suitable solvent to create a stationary liquid phase. The solvent penetrates the paper by capillary
action and, in passing over the sample spot, carries along with it the various components of the
sample.
What is thin layer chromatography (TLC)?
Thin layer chromatography (TLC) is a chromatographic technique used to separate the components
of a mixture using a thin stationary phase, thin-layer chromatography can be used to monitor the
progress of a reaction, identify compounds present in a given mixture and determine the purity of a
substance. This type of chromatography is the preferred method because it uses a more accurate
and high-quality material than paper chromatography. In thin layer chromatography the stationary
phase is a thin adsorbent material layer, usually silica gel or aluminium oxide, which is coated onto
an inert plate surface. The mobile phase travels up the plate by capillary forces and sample
components travel varying distances based on their differences for the stationary and mobile
phases. When the solvent reaches the top of the plate, the plate is removed from the chamber and
dried. The separated components appear as spots on the plate and the Rf (retention factor) of each
of the components can be assessed.
A: Paper chromatography of amino acids
, Equipment:
· Gas jar
· Chromatography paper
· Pencil
· Ruler
· Gloves
· Amino acid mixture (containing three unknown amino acids)
· Solvent
· Capillary tubing
· Ninhydrin spray
· Hair dryer or oven set at 110°
Method:
1. You are provided with an amino acid solution. The result is a solution of amino acids.
2. Wearing eye protection, place your gas jar in the fume cupboard and pour solvent in to a depth of
about 3cm. Place the lid on the gas jar and leave it in the fume cupboard so that the atmosphere
inside the gas jar becomes saturated with solvent fumes.
3. Wash and dry hands.
4. Take a piece of chromatography paper - it should be long enough for about 5mm to dip into the
solvent and the other end should extend out of the top of the gas jar by about 20mm. Handle the
paper as little as possible throughout the experiment.
5. Draw a faint pencil line about 40mm from one end of the chromatography paper.
6. Using the capillary tubing, draw up amino solution and place a tiny dot in the centre of the line.
7. Wait for it to dry.
8. Place another dot of amino acid solution on top of the first one using the capillary tubing and wait
for it to dry.
9. Repeat this at least six times letting it dry between applications until you have a very concentrated
dot of amino acid solution. Try to keep the dot as small as possible by being patient and letting it dry
fully between applications.
10. Carefully lower the paper into the gas jar, making sure it does not touch the sides and allow it to
just dip into the solvent. Fold the top of the paper over and replace the lid. Leave for several hours
until the solvent has moved to within 2cm of the top of the paper.
11. After several hours and once the solvent has moved to within 2cm of the top of the paper,
carefully remove the chromatography paper from the gas jar.
12. Without damaging the paper, draw another pencil line marking the point where the solvent
moved to and then hang the paper somewhere warm to dry.
Unit 2: Practical Scientific Procedures and Techniques
Learning Aim C: Undertake chromatographic techniques to identify components in mixtures
Matthew Howard (9360)
May 2022
Introduction: What is chromatography?
Chromatography can be described as the process of separating components into a mixture. To get
this process started, the mixture is dissolved into a substance called the mobile phase, this then
carries it towards the second substance which is called the stationary phase. The mobile phase may
be either a liquid or a gas, while the stationary phase is either a solid or a liquid. Different substances
will travel through the stationary phase at different speeds, this will then cause it to separate from
one another, this is how the results of chromatography are shown and collected. The travel times of
the substances is called the retention time which is used to find out the results of the experiment. By
altering the mobile phase and the stationary phase a wide variety of chromatographic methods have
been created, each serving a different purpose and ideal for different mixtures. Some of the most
common forms of chromatography are paper chromatography, thin layer chromatography, liquid
chromatography and gas chromatography.
What is paper chromatography?
Paper chromatography is an analytical method used to separate coloured substances by taking
advantage of their different rates of migration across sheets of paper. This chromatography
technique is most commonly used in a school setting now but it has been replaced in the laboratory
setting by thin layer chromatography, this is because it shows a greater separation of the
components in the mixture which makes it much easier to analyse. The method consists of applying
the test solution as a spot near one corner of a sheet of filter paper, the paper is initially applied with
a suitable solvent to create a stationary liquid phase. The solvent penetrates the paper by capillary
action and, in passing over the sample spot, carries along with it the various components of the
sample.
What is thin layer chromatography (TLC)?
Thin layer chromatography (TLC) is a chromatographic technique used to separate the components
of a mixture using a thin stationary phase, thin-layer chromatography can be used to monitor the
progress of a reaction, identify compounds present in a given mixture and determine the purity of a
substance. This type of chromatography is the preferred method because it uses a more accurate
and high-quality material than paper chromatography. In thin layer chromatography the stationary
phase is a thin adsorbent material layer, usually silica gel or aluminium oxide, which is coated onto
an inert plate surface. The mobile phase travels up the plate by capillary forces and sample
components travel varying distances based on their differences for the stationary and mobile
phases. When the solvent reaches the top of the plate, the plate is removed from the chamber and
dried. The separated components appear as spots on the plate and the Rf (retention factor) of each
of the components can be assessed.
A: Paper chromatography of amino acids
, Equipment:
· Gas jar
· Chromatography paper
· Pencil
· Ruler
· Gloves
· Amino acid mixture (containing three unknown amino acids)
· Solvent
· Capillary tubing
· Ninhydrin spray
· Hair dryer or oven set at 110°
Method:
1. You are provided with an amino acid solution. The result is a solution of amino acids.
2. Wearing eye protection, place your gas jar in the fume cupboard and pour solvent in to a depth of
about 3cm. Place the lid on the gas jar and leave it in the fume cupboard so that the atmosphere
inside the gas jar becomes saturated with solvent fumes.
3. Wash and dry hands.
4. Take a piece of chromatography paper - it should be long enough for about 5mm to dip into the
solvent and the other end should extend out of the top of the gas jar by about 20mm. Handle the
paper as little as possible throughout the experiment.
5. Draw a faint pencil line about 40mm from one end of the chromatography paper.
6. Using the capillary tubing, draw up amino solution and place a tiny dot in the centre of the line.
7. Wait for it to dry.
8. Place another dot of amino acid solution on top of the first one using the capillary tubing and wait
for it to dry.
9. Repeat this at least six times letting it dry between applications until you have a very concentrated
dot of amino acid solution. Try to keep the dot as small as possible by being patient and letting it dry
fully between applications.
10. Carefully lower the paper into the gas jar, making sure it does not touch the sides and allow it to
just dip into the solvent. Fold the top of the paper over and replace the lid. Leave for several hours
until the solvent has moved to within 2cm of the top of the paper.
11. After several hours and once the solvent has moved to within 2cm of the top of the paper,
carefully remove the chromatography paper from the gas jar.
12. Without damaging the paper, draw another pencil line marking the point where the solvent
moved to and then hang the paper somewhere warm to dry.
